Antibody-conjugates, i.e. antibodies conjugated to a molecule of interest via a linker, are known in the art. There is great interest in antibody-conjugates wherein the molecule of interest is a drug, for example a cytotoxic chemical. Antibody-drug-conjugates are known in the art, and consist of a recombinant antibody covalently bound to a cytotoxic chemical via a synthetic linker (S. C. Alley et al, Curr. Opin. Chem. Biol. 2010, 14, 529-537, incorporated by reference). The main objective of an antibody-drug-conjugate (ADC), also called immunotoxin, is to combine the high specificity of a monoclonal antibody for a tumor-associated antigen with the pharmacological potency of a “small” cytotoxic drug (typically 300 to 1,000 Da). Examples of ADCs include gemtuzumab ozogamicin (Mylotarg; anti-CD33 mAb conjugated to calicheamycin, Pfizer/Wyeth); brentuximab vedotin (SGN-35, Adcetris, a CD30-targeting ADC consisting of brentuximab, covalently linked to MMAE (monomethylauristatin), Seattle Genetics); trastuzumab-DM1 conjugate (T-DM1).
One advance in the field includes the emergence of extremely potent toxins, in particular taxanes, calicheamycins, maytansins, pyrrolobenzodiazepines, duocarmycins and auristatins. The low nanomolar to picomolar toxicity of these substances is a principal driver improvement over the earlier applied toxins. Another important technological advance involves the use of optimized linkers that are hydrolysable in the cytoplasm, resistant or susceptible to proteases, or resistant to multi-drug resistance efflux pumps that are associated with highly cytotoxic drugs.
ADCs known from the prior art are commonly prepared by conjugation of the linker-toxin to the side chain of antibody amino acid lysine or cysteine, by acylation or alkylation, respectively.
For lysines, conjugation takes place preferentially at lysine side chains with highest steric accessibility, the lowest pKa, or a combination thereof. Disadvantage of this method is that site-control of conjugation is low.
Better control of site-specificity is obtained by alkylation of cysteines, based on the fact that typically no free cysteines are present in an antibody, thereby offering the option of alkylating only those cysteines that are selectively liberated by a reductive step or specifically engineered into the antibody as free cysteines (as in so-called Thiomabs). Selective cysteine liberation by reduction is typically performed by treatment of whole antibody with a reducing agent (e.g. TCEP or DTT), leading to conversion of a disulfide bond into two free thiols (mostly in the antibody's hinge region). The liberated thiols are then alkylated with an electrophilic reagent, typically based on a maleimide attached to a linker-toxin, which generally proceeds fast and with high selectivity. With respect to engineering of an additional (free) cysteine into an antibody, enhanced site-control is attained with respect to the location of the added cysteine(s) and no reductive step is required, thereby ideally avoiding multiple disulfide bond cleavages and multiple alkylation. Also in this strategy alkylation of free cysteines is effected with maleimide chemistry, but full homogeneity is not attained.
At the same time, a disadvantage of ADCs obtained via alkylation with maleimides is that in general the resulting conjugates are unstable due to the reverse of alkylation, i.e. a retro-Michael reaction, thereby leading to release of linker-toxin from the antibody. Conjugation based on cysteine-maleimide alkylation is clearly not an ideal technology for developments of ADCs that preferably should not show premature release of toxin.
It is known in the art that azides (N3 groups, also referred to as azido groups) can undergo selective cycloaddition with terminal alkynes (copper-catalyzed) or with cyclic alkynes (by virtue of ring strain). The triazoles resulting from reaction with alkynes are not susceptible to hydrolysis or other degradation pathways. It is also known in the art that ketones can undergo selective conjugation with hydroxylamines or hydrazines, resulting in respectively oximes or hydrazones. Oximes and hydrazones are also relatively inert at neutral conditions but may undergo hydrolysis at lower pH.
Several methods for the introduction of an azide into a protein have been reviewed recently by van Delft et al., ChemBioChem. 2011, 12, 1309 (incorporated by reference): (a) chemoselective diazo transfer reaction, (b) enzymatic conversion, (c) expression in auxotrophic bacteria and (d) genetic encoding. Chemoselective diazo transfer reactions (a) have limited applicability for antibodies since these typically take place on a protein's N-terminus, whereas in an antibody the N-termini of both heavy and light chain are in the binding region of the antibody. Expression in auxotrophic bacteria (c) and genetic encoding (d) are, albeit powerful, very complex strategies for non-specialized laboratories and furthermore cannot be used for straightforward post-modification of existing recombinant antibodies and are therefore not generally applicable.
With respect to enzymatic conversion of proteins (b), several enzymes have been reported over the years capable of achieving such transformation with unnatural, azide-containing substrates, e.g. transglutaminase, lipoic acid ligase, sortase, FGE and others (reviewed in Bertozzi et al., Angew. Chem. Int. Ed. 2009, 48, 6974, incorporated by reference). Without exception, however, these enzymes require specific recognition sequences in the protein, which need to be specifically engineered, and conjugation may be restricted to the protein's termini.
A potentially versatile strategy that may be generally applicable to all monoclonal antibodies involves the specific enzymatic conjugation to the Fc-attached glycan, which is naturally present in all antibodies expressed in mammalian (or yeast) cell cultures. Several strategies based on this concept are known in the art, such as via oxidation of the terminal galactose or via transfer of (unnatural) sialic acid to the same galactose moiety. However, for ADC purpose such a strategy is suboptimal because glycans are always formed as a complex mixture of isoforms, which may contain different levels of galactosylation (G0, G1, G2) and therefore would afford ADCs with poor control of drug-antibody ratio (DAR, see below).
FIG. 1 shows an antibody comprising a glycan on each heavy chain. These glycans are present in different isoforms with respect to galactosylation (G0, G1 and G2) and fucosylation (G0F, G1F and G2F).
In WO 2007/133855 (University of Maryland Biotechnology Institute), incorporated by reference herein, a chemoenzymatic method for the preparation of a homogeneous glycoprotein or glycopeptide is disclosed, involving a two-stage strategy entailing first trimming of the near-complete glycan tree (under the action of endo A or endo H) leaving only the core N-acetylglucosamine (GlcNAc) moiety (the so-called GlcNAc-protein), followed by a reglycosylation event wherein, in the presence of a catalyst comprising endoglycosidase (ENGase), an oligosaccharide moiety is transferred to the GlcNAc-protein to yield a homogeneous glycoprotein or glycopeptide. A strategy for azide-functionalized glycoproteins is disclosed, wherein a GlcNAc-protein is reacted in the presence of ENGase with a tetrasaccharide oxazoline containing two 6-azidomannose moieties, thereby introducing two azides simultaneously in the glycan. The azide-functionalized glycoprotein may then be catalytically reacted in a “click chemistry” cycloaddition reaction, in the presence of a catalyst (e.g. a Cu(I) catalyst) with a terminal alkyne bearing a functional moiety X of interest. No actual examples of said click chemistry are disclosed.
In J. Am. Chem. Soc. 2008, 130, 13790, incorporated by reference herein, Wang et al. disclose an efficient double attachment of a terminal alkyne-modified trisaccharide to bis-azidomodified ribonuclease B by copper-catalyzed click chemistry.
In J. Am. Chem. Soc. 2012, 134, 8030, incorporated by reference herein, Davis et al. disclose the transfer of oligosaccharide oxazolines on a core-fucosylated as well as nonfucosylated core-GlcNAc-Fc domain of intact antibodies, in the presence of glycosynthase EndoS.
In J. Am. Chem. Soc. 2012, 134, 12308, incorporated by reference herein, Wang et al. disclose the transfer of a tetrasaccharide oxazoline containing two 6-azidomannose moieties on core-fucosylated as well as nonfucosylated core-GlcNAc-Fc domain of intact antibodies (Rituximab) in the presence of glycosynthase mutants EndoS-D233A and EndoS-D233Q. This process is shown schematically in FIG. 2, and results in an antibody comprising four azido groups. The subsequent conjugation of the azido-modified IgG with click chemistry is mentioned but not disclosed.
However, a disadvantage of the glycosynthase strategies disclosed in WO 2007/133855, J. Am. Chem. Soc. 2012, 134, 8030 and J. Am. Chem. Soc. 2012, 134, 12308 is the lengthy and complex synthesis of the required azido-containing oligosaccharide oxazolines. In addition, the azido-containing oligosaccharide oxazolines comprise two azido groups. To date, it has not been shown whether this process may be suitable for the introduction of only one azido group on an antibody glycan.
Qasba et al. disclose in J. Biol. Chem. 2002, 277, 20833, incorporated by reference herein, that mutant galactosyltransferases GalT(Y289L), GalT(Y289I) and GalT(Y289N) can enzymatically attach GalNAc to a non-reducing GlcNAc sugar ((3-benzyl-GlcNAc).
WO 2004/063344 (National Institutes of Health), incorporated by reference herein, discloses mutant galactosyltransferases GalT(Y289L), GalT(Y289I) and GalT(Y289N). A process is disclosed wherein complex glycans such as those on monoclonal antibodies (Rituxan, Remicade, Herceptin) are first converted into G0 glycans on antibodies by treatment with galactosidase, in order to remove all chain-terminal galactose. These G0 antibodies are subsequently subjected to GalNAc-UDP in the presence of GalT(Y289L), leading to antibodies with significantly more homogeneous glycan structures.
Qasba et al. disclose in Bioconjugate Chem. 2009, 20, 1228, incorporated by reference herein, that the process disclosed in WO 2004/063344 also proceeds for non-natural GalNAc-UDP variants substituted on the N-acetyl group. β-Galactosidase treated monoclonal antibodies having a G0 glycoform are fully galactosylated to the G2 glycoform after transfer of a galactose moiety comprising a C2-substituted azidoacetamido moiety (GalNAz) to the terminal GlcNAc residues of the glycan, leading to tetraazido-substituted antibodies, i.e. two GalNAz moieties per heavy chain. This process is shown schematically in FIG. 3. The conjugation of said tetraazido-substituted antibodies to a molecule of interest was not disclosed. The transfer of a galactose moiety comprising a C2-substituted keto group (C2-keto-Gal) to the terminal GlcNAc residues of a G0 glycoform glycan, as well as the linking of C2-keto-Gal to aminooxy biotin, is also disclosed. In all cases, GalT(Y289L) mutant is used for the transfer of GalNAc-UDP (or GalNAz-UDP) to the antibodies, but the use of mutants GalT(Y289N) or GalT(Y289I) is not disclosed.
A disadvantage of the method disclosed in WO 2004/063344 and Bioconjugate Chem. 2009, 20, 1228 is that conjugation of the tetraazido-substituted antibodies to a molecule of interest would lead to an antibody-conjugate with typically two molecules of interest per glycan (provided that said conjugation would proceed with complete conversion). In some cases, for example when the molecule of interest is a lipophilic toxin, the presence of too many molecules of interest per antibody is undesired since this may lead to aggregate formation (BioProcess International 2006, 4, 42-43, incorporated by reference).
WO 2007/095506 and WO 2008/029281 (Invitrogen Corporation), incorporated by reference herein, disclose a method of forming a glycoprotein conjugate wherein the glycoprotein is contacted with UDP-GalNAz in the presence of GalT(Y289L) mutant, leading to the incorporation of GalNAz at a terminal non-reducing GlcNAc of an antibody carbohydrate. Subsequent copper-catalyzed click chemistry with a terminal alkyne or Staudinger ligation can then be used to conjugate a reporter molecule, solid support or carrier molecule to the attached azide moiety. WO 2007/095506 and WO 2008/029281 further disclose that if no terminal GlcNAc sugars are present on the antibody, Endo H, Endo A or Endo M enzyme may be used to generate a truncated chain which terminates with one N-acetylglucosamine residue.
Antibody-conjugates known in the art generally suffer from several disadvantages. For antibody drug-conjugates, a measure for the loading of the antibody with a toxin is given by the drug-antibody ratio (DAR), which gives the average number of active substance molecules per antibody. However, the DAR does not give any indication regarding the homogeneity of such ADC.
Processes for the preparation of an antibody-conjugate known from the prior art generally result in a product with a DAR between 1.5 and 4, but in fact such a product comprises a mixture of antibody-conjugates with a number of molecules of interest varying from 0 to 8 or higher. In other words, antibody-conjugates known from the prior art generally are formed with a DAR with high standard deviation.
For example, gemtuzumab ozogamicin is a heterogeneous mixture of 50% conjugates (0 to 8 calicheamycin moieties per IgG molecules with an average of 2 or 3, randomly linked to solvent exposed lysyl residues of the antibody) and 50% unconjugated antibody (Bross et al., Clin. Cancer Res. 2001, 7, 1490; Labrijn et al., Nat. Biotechnol. 2009 27, 767, both incorporated by reference). But also for brentuximab vedotin, T-DM1, and other ADCs in the clinic, it is still uncontrollable exactly how many drugs are attaching to any given antibody (drug-antibody ratio, DAR) and the ADC is obtained as a statistical distribution of conjugates. Whether the optimal number of drugs per antibody is for example two, four or more, attaching them in a predictable number and in predictable locations through site-specific conjugation with a narrow standard deviation is still problematic.